The role of Cra in regulating acetate excretion and osmotic tolerance in E. coli K-12 and E. coli B at high density growth

<p>Abstract</p> <p>Background</p> <p><it>E. coli </it>B (BL21), unlike <it>E.coli </it>K-12 (JM109) is insensitive to glucose concentration and, therefore, grows faster and produces less acetate than <it>E. coli </it>K-12, especially when growing to high cell densities at high glucose concentration. By performing genomic analysis, it was demonstrated that the cause of this difference in sensitivity to the glucose concentration is the result of the differences in the central carbon metabolism activity. We hypothesized that the global transcription regulator Cra (FruR) is constitutively expressed in <it>E. coli </it>B and may be responsible for the different behaviour of the two strains. To investigate this possibility and better understand the function of Cra in the two strains, <it>cra </it>- negative <it>E. coli </it>B (BL21) and <it>E. coli </it>K-12 (JM109) were prepared and their growth behaviour and gene expression at high glucose were evaluated using microarray and real-time PCR.</p> <p>Results</p> <p>The deletion of the <it>cra </it>gene in <it>E. coli </it>B (BL21) minimally affected the growth and maximal acetate accumulation, while the deletion of the same gene in <it>E.coli </it>K-12 (JM109) caused the cells to stop growing as soon as acetate concentration reached 6.6 g/L and the media conductivity reached 21 mS/cm. <it>ppsA </it>(gluconeogenesis gene), <it>aceBA </it>(the glyoxylate shunt genes) and <it>poxB </it>(the acetate producing gene) were down-regulated in both strains, while <it>acs </it>(acetate uptake gene) was down-regulated only in <it>E.coli </it>B (BL21). These transcriptional differences had little effect on acetate and pyruvate production. Additionally, it was found that the lower growth of <it>E. coli </it>K-12 (JM109) strain was the result of transcription inhibition of the osmoprotectant producing <it>bet </it>operon (<it>betABT</it>).</p> <p>Conclusions</p> <p>The transcriptional changes caused by the deletion of <it>cra </it>gene did not affect the activity of the central carbon metabolism, suggesting that Cra does not act alone; rather it interacts with other pleiotropic regulators to create a network of metabolic effects. An unexpected outcome of this work is the finding that <it>cra </it>deletion caused transcription inhibition of the <it>bet </it>operon in <it>E. coli </it>K-12 (JM109) but did not affect this operon transcription in <it>E. coli </it>B (BL21). This property, together with the insensitivity to high glucose concentrations, makes this the <it>E. coli </it>B (BL21) strain more resistant to environmental changes.</p

Engineering alternative butanol production platforms in heterologous bacteria

Alternative microbial hosts have been engineered as biocatalysts for butanol biosynthesis. The butanol synthetic pathway of Clostridium acetobutylicum was first re-constructed in Escherichia coli to establish a baseline for comparison to other hosts. Whereas polycistronic expression of the pathway genes resulted in the production of 34 mg/L butanol, individual expression of pathway genes elevated titers to 200 mg/L. Improved titers were achieved by co-expression of Saccharomyces cerevisiae formate dehydrogenase while overexpression of E. coli glyceraldehyde 3-phosphate dehydrogenase to elevate glycolytic flux improved titers to 580 mg/L. Pseudomonas putida and Bacillus subtilis were also explored as alternative production hosts. Polycistronic expression of butanol biosynthetic genes yielded butanol titers of 120 and 24 mg/L from P. putida and B. subtilis, respectively. Production in the obligate aerobe P. putida was dependent upon expression of bcd-etfAB. These results demonstrate the potential of engineering butanol biosynthesis in a variety of heterologous microorganisms, including those cultivated aerobically.Synthetic Biology Engineering Research CenterNational Science Foundation (Grant no. 0540879)Massachusetts Institute of Technology. Energy Initiative (Grant no. 6917278)Natural Sciences and Engineering Research Council of CanadaKorea Research Foundation (Grant

Mechanically ventilated COVID-19 patients admitted to the intensive care unit in the United States with or without respiratory failure secondary to COVID-19 pneumonia: a retrospective comparison of characteristics and outcomes

Background There is increasing heterogeneity in the clinical phenotype of patients admitted to the intensive care unit (ICU) with coronavirus disease 2019 (COVID-19,) and reasons for mechanical ventilation are not limited to COVID pneumonia. We aimed to compare the characteristics and outcomes of intubated patients admitted to the ICU with the primary diagnosis of acute hypoxemic respiratory failure (AHRF) from COVID-19 pneumonia to those patients admitted for an alternative diagnosis. Methods Retrospective cohort study of adults with confirmed SARS-CoV-2 infection admitted to nine ICUs between March 18, 2020, and April 30, 2021, at an urban university institution. We compared characteristics between the two groups using appropriate statistics. We performed logistic regression to identify risk factors for death in the mechanically ventilated COVID-19 population. Results After exclusions, the final sample consisted of 319 patients with respiratory failure secondary to COVID pneumonia and 150 patients intubated for alternative diagnoses. The former group had higher ICU and hospital mortality rates (57.7% vs. 36.7%, P<0.001 and 58.9% vs. 39.3%, P<0.001, respectively). Patients with AHRF secondary to COVID-19 pneumonia also had longer ICU and hospital lengths-of-stay (12 vs. 6 days, P<0.001 and 20 vs. 13.5 days, P=0.001). After risk-adjustment, these patients had 2.25 times higher odds of death (95% confidence interval, 1.42–3.56; P=0.001). Conclusions Mechanically ventilated COVID-19 patients admitted to the ICU with COVID-19-associated respiratory failure are at higher risk of hospital death and have worse ICU utilization outcomes than those whose reason for admission is unrelated to COVID pneumonia

Paired termini stabilize antisense RNAs and enhance conditional gene silencing in Escherichia coli

Reliable methods for conditional gene silencing in bacteria have been elusive. To improve silencing by expressed antisense RNAs (asRNAs), we systematically altered several design parameters and targeted multiple reporter and essential genes in Escherichia coli. A paired termini (PT) design, where flanking inverted repeats create paired dsRNA termini, proved effective. PTasRNAs targeted against the ackA gene within the acetate kinase-phosphotransacetylase operon (ackA-pta) triggered target mRNA decay and a 78% reduction in AckA activity with high genetic penetrance. PTasRNAs are abundant and stable and function through an RNase III independent mechanism that requires a large stoichiometric excess of asRNA. Conditional ackA silencing reduced carbon flux to acetate and increased heterologous gene expression. The PT design also improved silencing of the essential fabI gene. Full anti-fabI PTasRNA induction prevented growth and partial induction sensitized cells to a FabI inhibitor. PTasRNAs have potential for functional genomics, antimicrobial discovery and metabolic flux control

High-flux isobutanol production using engineered Escherichia coli: a bioreactor study with in situ product removal

Promising approaches to produce higher alcohols, e.g., isobutanol, using Escherichia coli have been developed with successful results. Here, we translated the isobutanol process from shake flasks to a 1-L bioreactor in order to characterize three E. coli strains. With in situ isobutanol removal from the bioreactor using gas stripping, the engineered E. coli strain (JCL260) produced more than 50 g/L in 72 h. In addition, the isobutanol production by the parental strain (JCL16) and the high isobutanol-tolerant mutant (SA481) were compared with JCL260. Interestingly, we found that the isobutanol-tolerant strain in fact produced worse than either JCL16 or JCL260. This result suggests that in situ product removal can properly overcome isobutanol toxicity in E. coli cultures. The isobutanol productivity was approximately twofold and the titer was 9% higher than n-butanol produced by Clostridium in a similar integrated system

Identification of Lactoferricin B Intracellular Targets Using an Escherichia coli Proteome Chip

Lactoferricin B (LfcinB) is a well-known antimicrobial peptide. Several studies have indicated that it can inhibit bacteria by affecting intracellular activities, but the intracellular targets of this antimicrobial peptide have not been identified. Therefore, we used E. coli proteome chips to identify the intracellular target proteins of LfcinB in a high-throughput manner. We probed LfcinB with E. coli proteome chips and further conducted normalization and Gene Ontology (GO) analyses. The results of the GO analyses showed that the identified proteins were associated with metabolic processes. Moreover, we validated the interactions between LfcinB and chip assay-identified proteins with fluorescence polarization (FP) assays. Sixteen proteins were identified, and an E. coli interaction database (EcID) analysis revealed that the majority of the proteins that interact with these 16 proteins affected the tricarboxylic acid (TCA) cycle. Knockout assays were conducted to further validate the FP assay results. These results showed that phosphoenolpyruvate carboxylase was a target of LfcinB, indicating that one of its mechanisms of action may be associated with pyruvate metabolism. Thus, we used pyruvate assays to conduct an in vivo validation of the relationship between LfcinB and pyruvate level in E. coli. These results showed that E. coli exposed to LfcinB had abnormal pyruvate amounts, indicating that LfcinB caused an accumulation of pyruvate. In conclusion, this study successfully revealed the intracellular targets of LfcinB using an E. coli proteome chip approach

ANALISIS DAN PERANCANGAN SISFO PERSEDIAAN BAHAN BAKU PADA PT SUMBER INTI ANEKA SEMPANA

ANALISIS DAN PERANCANGAN SISFO PERSEDIAAN BAHAN BAKU PADA PT SUMBER INTI ANEKA SEMPAN

Double Rainbows

Double rainbows.https://trace.tennessee.edu/utiaphoto_2014/1618/thumbnail.jp

Web applications - New mobile service paradigm

The explosion of mobile applications both in number and variety raises the need of shedding light on their architecture, composition and quality. Indeed, it is crucial to understand which mobile application paradigm fits better to what type of application and usage. Such understanding has direct consequences on the user experience, the development cost and sale revenues of mobile apps. In this thesis, we identify four main mobile application paradigms and evaluate them from the viewpoints of developers, users and service providers. To ensure objectivity and accuracy we start by defining high level criteria and then breaking down into finer-grained criteria. After a theoretical evaluation an implementation was carried out as a practical verification to ensure that the method adopted in analysis and evaluation is trusted and applicable. The selected application is object recognition app, which is both exciting and challenging to develop